The particular structural arrangement of chaperonins probably contributes to their ability to assist in the folding of proteins. The interaction of the oligomeric bacterial chaperonin GroEL and its cochaperonin, GroES, in the presence of adenosine diphosphate (ADP) forms an asymmetric complex. However, in the presence of adenosine triphosphate (ATP) or its nonhydrolyzable analogs, symmetric complexes were found by electron microscopy and image analysis. The existence of symmetric chaperonin complexes is not predicted by current models of the functional cycle for GroE-mediated protein folding. Because complete folding of a nonnative substrate protein in the presence of GroEL and GroES only occurs in the presence of ATP, but not with ADP, the symmetric chaperonin complexes formed during the GroE cycle are proposed to be functionally significant.

The GroE chaperones of Escherichia coli assist protein folding under physiological and heat shock conditions in an ATP-dependent way. Although a number of details of assisted folding have been elucidated, the molecular mechanism of the GroE cycle remains unresolved. Here we present an experimental system that allows the direct analysis of the GroE-mediated folding cycle under stringent conditions. We demonstrate that the GroE proteins efficiently catalyze the folding of kinetically trapped folding intermediates of a mutant of maltose-binding protein (MBP Y283D) in an ATP-dependent way. GroES plays a key role in this reaction cycle, accelerating the folding of the substrate protein MBP Y283D up to 50-fold. Interestingly, catalysis of the folding reaction requires the formation of symmetrical footballshaped GroEL⅐GroES 2 particles and the intermediate release of the nonnative protein from the chaperone complex. Our results show that, in the presence of GroES, the complex architecture of the GroEL toroids allows maintenance of two highly interregulated rings simultaneously active in protein folding.Chaperonins play an important role in maintaining protein integrity under physiological as well as under heat shock conditions (1-4). Forming tight complexes with folding intermediates, they prevent aggregation and assist the folding of polypeptides that would not reach the native state in a spontaneous reaction, due to the nonpermissive conditions of the cellular environment (5-7).The GroE chaperone complex comprises two different proteins. GroEL, the protein binding component, consists of two stacked rings with seven identical subunits each (8). Each ring contains a central cavity in which nonnative protein can be accommodated (9-11). The co-chaperonin GroES is a seven-membered ring (12)(13)(14). Binding of either one or two GroES rings to GroEL leads to the formation of asymmetric bullet-shaped or symmetric football-shaped particles, as identified by electron microscopy (15-17). The significance of football-shaped particles as obligate or facultative intermediates in the ATPase cycle as well as in the coupled protein folding cycle are still unclear (18-25). However, it was shown that, in principle, folding can occur in the cavity of GroEL covered by GroES (cis-bullet) in a single turnover event (23,24,26).Although GroE has been shown to be able to rescue aggregated protein, thereby enhancing the yield and the kinetics of refolding (27, 28), and to reshuffle trapped folding intermediates to a productive folding pathway via an unfolding event (29,30), until now no quantitative experimental system, describing the way GroE influences folding, has been reported, and the underlying mechanisms remain to be elucidated.Here we used the temperature-sensitive folding mutant MBP Y283D, a 40-kDa periplasmic protein of Escherichia coli, as a substrate protein for GroE. This maltose-binding protein (MBP) variant provides the opportunity to directly study the effects of GroE on a slow folding species that does not aggregate b...

The Escherichia coli GroE chaperones assist protein folding under conditions where no spontaneous folding occurs. To achieve this, the cooperation of GroEL and GroES, the two protein components of the chaperone system, is an essential requirement. While in many cases GroE simply suppresses unspeci®c aggregation of non-native proteins by encapsulation, there are examples where folding is accelerated by GroE.Using maltose-binding protein (MBP) as a substrate for GroE, it had been possible to de®ne basic requirements for catalysis of folding. Here, we have analyzed key steps in the interaction of GroE and the MBP mutant Y283D during catalyzed folding. In addition to high temperature, high ionic strength was shown to be a restrictive condition for MBP Y283D folding. In both cases, the complete GroE system (GroEL, GroES and ATP) compensates the deceleration of MBP Y283D folding. Combining kinetic folding experiments and electron microscopy of GroE particles, we demonstrate that at elevated temperatures, symmetrical GroE particles with GroES bound to both ends of the GroEL cylinder play an important role in the ef®cient catalysis of MBP Y283D refolding. In principle, MBP Y283D folding can be catalyzed during one encapsulation cycle. However, because the commitment to reach the native state is low after only one cycle of ATP hydrolysis, several interaction cycles are required for catalyzed folding.

Enolase (2-phospho-D-glycerate hydrolase; EC 4.2.1.11) from the hyperthermophilic bacterium Thermotoga maritima was purified to homogeneity. The N-terminal 25 amino acids of the enzyme reveal a high degree of similarity to enolases from other sources. As shown by sedimentation analysis and gel-permeation chromatography, the enzyme is a 345-kDa homoctamer with a subunit molecular mass of 48 +/- 5 kDa. Electron microscopy and image processing yield ring-shaped particles with a diameter of 17 nm and fourfold symmetry. Averaging of the aligned particles proves the enzyme to be a tetramer of dimers. The enzyme requires divalent cations in the activity assay, Mg2+ being most effective. The optimum temperature for catalysis is 90 degrees C, the temperature dependence yields a nonlinear Arrhenius profile with limiting activation energies of 75 kJ mol-1 and 43 kJ mol-1 at temperatures below and above 45 degrees C. The pH optimum of the enzyme lies between 7 and 8. The apparent Km values for 2-phospho-D-glycerate and Mg2+ at 75 degrees C are 0.07 mM and 0.03 mM; with increasing temperature, they are decreased by factors 2 and 30, respectively. Fluoride and phosphate cause competitive inhibition with a Ki of 0.14 mM. The enzyme shows high intrinsic thermal stability, with a thermal transition at 90 and 94 degrees C in the absence and in the presence of Mg2+.

Lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has been functionally expressed in Escherichia coli. As shown by gel-permeation chromatography, dynamic light scattering, and ultracentrifugation, the recombinant protein forms homotetrameric and homooctameric assemblies with identical spectral properties and a common subunit molecular mass (35 kDa). Dynamic light scattering and sedimentation equilibrium experiments proved that both species are monodisperse, thus excluding their interconversion in the given ranges of concentration (0.02 -50 mg/ml) and temperature (20-SOOC). Rechromatography confirms this finding : the octamer does not dissociate at low enzyme concentrations, nor do tetramers dimerize at the given upper limit of concentration. Renaturation of pure tetramers or octamers after preceding guanidine denaturation leads to redistribution of the two species ; increased temperature favors octamer formation.Thermal analysis and denaturation by chaotropic agents do not allow the free energies of stabilization of the two forms to be quantified, because heat coagulation and kinetic partitioning between reconstitution and aggregation cause irreversible side reactions. Guanidine denaturation of the octamer leads to a highly cooperative dissociation to tetramers which subsequently dissociate and unfold to yield metastable dimers and, finally, fully unfolded monomers. Evidently, there is no tight coupling of the two tetramers within the stable octameric quaternary structure. Electron microscopy clearly corroborates this conclusion : image processing shows that the dumb-bell-shaped octamer is made up of two tetramers connected via surface contacts without significant changes in the dimensions of the constituent parts.Keywords: hyperthermophiles ; lactate dehydrogenase; quaternary structure ; stability ; Thermotoga muritimu.Lactate dehydrogenases (LDH) are dimeric or tetrameric enzymes that require their native quaternary structure for catalysis (Jaenicke, 1974). As has been shown by denaturation-renaturation experiments, using domain fragments and intact subunits of porcine skeletal muscle LDH, tertiary and quaternary interactions provide increments of stability which in toto yield the exceedingly high value for the free energy of stabilization observed for the enzyme (Miiller et al., 1982;Pfeil, 1986; Opitz et al., 1987;Jaenicke, 1991). This supports the general view that protein stability is accomplished by the cumulative effect of covalent and non-covalent bonds at the various levels of the hierarchy of protein structure (Vita et al.. 1989;Jaenicke, 1996).There are various enzymes from thermophilic (and hyperthermophilic) organisms that exhibit anomalously high states of association, which suggests thermal adaptation of proteins to be attributable to higher states of subunit assembly. For example, pyruvate dehydrogenase from Bacillus stearothermophilus (To,, ~6 5°C ) contains four times as many polypeptide chains comCorrespondence ta R. Jaenicke, lnstitut